ABSTRACT Mutations of oncogenes and tumor suppressor genes transform normal hematopoietic stem cells (HSC) into leukemic stem cells (LSC) in a multi-step process. As the first step of leukemogenesis, the ?pre-leukemia? mutations dys-regulate HSC functions to promote clonal expansion by increasing proliferation, competitiveness and self-renewal. Pre-LSCs are further transformed into LSCs by additional mutations. Targeting pre-LSCs and LSCs has the potential to eliminate leukemia. Although many oncogenes and tumor suppressor genes have been proposed to transform normal HSCs to LSCs, very little is known about what signaling mechanism underlies this transformation to allow development of therapies to target pre-LSCs and LSCs. The long term goal of our research is to identify signaling mechanisms through which oncogenes and tumor suppressor genes transform normal HSCs into LSCs. Clonal expansion has been difficult to recapitulate experimentally because mutations that drive HSCs into cycling often lead to reduced HSC self-renewal and HSC depletion. We recently found that an oncogenic Nras mutation commonly found in human leukemias, G12D, dys-regulates HSCs and transform them into pre-LSC with increased proliferation, competitiveness and self-renewal. The objective of this proposal is to identify the signaling mechanism underlying this Nras induced transformation. Based on our preliminary results, our central hypothesis is that JAK2/STAT5 signaling is critical in the Nras induced transformation to pre-LSCs and further mediates transformation to LSCs. We will test this hypothesis by 1) Identify the mechanism by which NrasG12D activates STAT5 to dys-regulate HSC functions; 2) Define the role of JAK2 in NrasG12D induced HSC dys-regulation and 3) determine the role of JAK2/STAT5 signaling in fully transformed LSCs. The approach is innovative because 1) it takes advantage of our newly developed clonal expansion model; 2) it combines mouse genetic analysis and cellular and biochemical analysis on rare populations to investigate signaling mechanisms in pre-LSCs and LSCs, and 3) it evaluates the role of non-canonical Ras effectors that may be responsible for the effects of oncogenic Ras in a population highly relevant to leukemogenesis. The proposed research is significant because it is expected to advance knowledge of the signaling mechanisms underlying the transformation from normal HSCs to LSCs, and therefore to inform novel therapeutic intervention that will target pre-LSCs and LSCs to eradicate leukemia.